Device for providing a means for internal calibration in an electrochemical sensor

ABSTRACT

Internally calibrated pH and other analyte sensors based on redox agents provide more accurate results when the redox active reference agent is in a constant chemical environment, yet separated from the solution being analyzed in such a way as to maintain electrical contact with the sample. Room temperature ionic liquids (RTIL) can be used to achieve these results when used as a salt bridge between the reference material and the sample being analyzed. The RTIL provides the constant chemical environment and ionic strength for the redox active material (RAM) and provides an electrolytic layer that limits or eliminates direct chemical interaction with the sample. A broad range of RAMs can be employed in a variety of configurations in such “Analyte Insensitive Electrode” devices.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/255,224, filed Nov. 17, 2011 and titled DEVICE FOR PROVIDING A MEANSFOR INTERNAL CALIBRATION IN AN ELECTROCHEMICAL SENSOR, which applicationis a United States National Stage application of InternationalApplication No. PCT/US2010/026842, filed Mar. 10, 2010 and claimingpriority from U.S. Provisional Application No. 61/158,849, filed Mar.10, 2009, all of which are incorporated herein by reference.

BACKGROUND

The present invention provides methods and devices for measuring theconcentration of an analyte in solution and relates to the field ofchemistry.

Most conventional pH sensors on the market today utilize an ionsensitive glass bulb sensitive to pH and an internal referenceelectrode. The reference electrode is usually a chloridized silver(Ag|AgCl) wire immersed in potassium chloride (KCl) gel or liquid andseparated from the sample being analyzed via a porous frit, as shown inthe schematic in FIG. 1. Both the use of an internal reference electrodeand the necessity for the inclusion of a porous frit impair theoperation of these conventional glass pH probes due to problems withdrift caused by changes in the reference electrode potential and foulingor blocking of the frit. Thus, these conventional pH probes requireconstant recalibration, the electrodes must be stored in a KCl solutionto keep the porous frit from drying out, and the fragile glass membranerenders these probes unsuitable for many applications where pHmeasurement is required under conditions of high temperature orpressure.

Effort has been made to improve the function of the reference electrodeby, for instance, modification of the electrode-analyte interface (seeU.S. Pat. No. 7,276,142) or replacement of the typically heterogeneousredox couple (e.g. calomel or silver/silver chloride) with a homogeneousredox couple (e.g. iodide/triiodide) (see U.S. Pat. No. 4,495,050).These changes are based on the extension of the potentiometric referenceelectrode concept wherein the conventional reference electrode (CRE)typically comprises two halves of a redox couple in contact with anelectrolyte of fixed ionic composition and ionic strength. Because bothhalves of the redox couple are present and the composition of all thespecies involved is fixed, the system is maintained at equilibrium andthe potential drop (i.e. the measured voltage) across theelectrode-electrolyte interface of the conventional reference electrodeis then thermodynamically fixed and constant. The function of thereference electrode is then to provide a fixed potential to which othermeasurements, such as pH, may be compared.

While these conventional reference electrodes provide a stablepotential, they suffer from many disadvantages. One disadvantage is theneed for an electrolyte of fixed and known ionic composition and ionicstrength, because any change in ionic composition or strength willresult in a shift in equilibrium of the redox couple, therebycompromising the stability of the constant potential of the electrode.To preclude a change in electrolyte composition, the redox system andelectrolyte are typically isolated from the sample under study via aporous frit or small aperture. This isolation introduces an additionaldisadvantage to the conventional reference electrode, namely thepropensity for the frit or aperture to clog, rendering the electrodeuseless. These disadvantages are exacerbated by the fact that theelectrolyte is typically an aqueous solution of high salt concentration,resulting in the requirement that the electrode frit or aperture must bekept wet to avoid clogging due to salt precipitation.

A remarkable advance in pH sensor technology is the solid stateinternally calibrated pH sensor comprised of two redox-active pHsensitive agents (anthraquinone (AQ) and 9,10-phenanthrenequinone (PAQ))and one pH insensitive redox agent (e.g., Ferrocene (Fc)); see PCTPatent Publication Nos. 2005/066618 and 2007/034131 and GB PatentPublication No. 2409902. In such sensors, all three redox agents may bemixed together with multiwalled carbon nanotubes (MWCNT), graphitepowder and epoxy, and the resulting admixture cured and formed intosolid sensors. When a voltage sweep is applied to the sensor and theresultant current measured (using square wave voltammetry, for example),one observes three peaks: one peak for each of the three redox agents.

In these solid state internally calibrated pH sensors, the pHinsensitive peak (due to Fc) should ideally be constant and independentof pH or ionic species in solution and should not drift over time. TheAQ and PAQ peaks should ideally vary their position on the voltage sweepin a predictable fashion depending on the pH of the solution beingmeasured. Finally, the positions of the pH sensitive peaks, whencompared to the position of the pH insensitive peak, allow the solutionpH to be deduced by comparing those values to a calibration table. Forthis system to have the greatest accuracy and so have the greatest scopeof application, the pH insensitive peak must be stable over time, andits peak position must be unaffected by varying solution compositions.Otherwise, the accuracy of the system is compromised. Unfortunately,most if not all pH insensitive redox agents appear to be affectedunsuitably by different ions and exhibit significant drift or shifts inpeak position. This problem is also present in other sensors thatrespond to analytes other than pH. Accordingly, there remains a need inthe art for materials and methods for making internally calibrated pHand other analyte sensors based on redox agents.

The present invention meets this need.

SUMMARY OF THE INVENTION

The present invention arises in part from the discoveries that (i) thepH insensitive material, or more generally, the analyte insensitivematerial (AIM), must be maintained in a constant chemical environment,yet separated from the solution being analyzed in such a way as tomaintain electrical contact with the sample being analyzed; (ii) anelectrolytic layer, which can be composed of, for example and withoutlimitation, room temperature ionic liquids (RTIL) or other ionic liquidsor liquids with sufficient ionic strength, can be used to achieve thedesired results when used as a salt bridge between the AIM and thesample being analyzed; and (iii) with such an electrolytic layer, ananalyte sensitive material (ASM) can be used in place of or in additionto an AIM, because the ASM is converted functionally into an AIM when asuitable electrolytic layer is employed. The electrolytic layer (e.g.composed of an RTIL or other suitable material, as described herein)provides the constant chemical environment and ionic strength for theAIM (or ASM) and provides a layer that limits or eliminates directchemical interaction with the sample being analyzed. A broad range ofredox active materials can be employed in a variety of configurations inaccordance with the methods and in the “analyte insensitive electrodes”(AIEs) of this invention and devices containing them.

The present invention provides a variety of AIEs for use in theinternally calibrated pH and other analyte sensors based on redox agentsprovided by the invention. The schematic shown in FIG. 2 providesillustrative embodiments of this aspect of the invention. In thatfigure, the oval dots represent the redox active material. Additionalembodiments are also provided and will be apparent to one of skill inthe art upon consideration of this disclosure; for example, in someembodiments, the electrolytic layer (e.g. RTIL or other material) is ina porous structure or “conductive physical barrier”, which serves tolimit direct chemical interaction and enhance electronic communicationbetween the sample test solution and the redox active material.

A wide variety of redox active materials (e.g. AIMs or ASMs) can be usedand placed in the different configurations of the various embodiments ofthe sensors of the invention, depending on the application for which adevice is intended and the solubility and/or other characteristics ofthe redox active material. For example, redox active materials that havebeen tested and demonstrated to be useful in the methods and devices ofthe invention are shown in FIG. 3.

Thus, the invention relates to compositions, devices, electrodes,sensors, systems, and methods useful for detecting the presence of ananalyte or measuring the analyte concentration in a sample. In oneaspect, the invention provides a means of continuous internalself-calibration for such measurements. In one aspect, the inventionprovides a substantially analyte insensitive electrode (AIE), and in oneembodiment, this electrode employs a substantially analyte insensitiveredox active material (AIM) to generate a predictable analyteinsensitive signal, while in another embodiment, this electrode employsa substantially analyte sensitive redox active material (ASM) togenerate a predictable analyte insensitive signal. In another aspect,the invention provides a voltammetric or amperometric analyte sensorsystem in which an AIE of the invention generates a signal that iscompared with an analyte sensitive signal from an analyte sensitiveelectrode (ASE) to provide a means for continuous internalself-calibration.

The present invention thus meets the need for an AIE capable ofgenerating a substantially analyte insensitive signal in response to theapplication of an electrical stimulus applied to the sample beinganalyzed in the course of making voltammetric or amperometricmeasurements of analyte concentration in the sample. The electrodes ofthe invention provide a predictable signal useful as an internalstandard (in other words, a standard internal to the system) with whichan analyte sensitive signal may be continuously compared, and thereforepermit greater accuracy and reproducibility in determining analyteconcentration.

The present invention therefore provides an AIE that can be used in anelectrochemical analyte sensing device that is capable of generating asubstantially analyte-insensitive electrical response when an electricalstimulus is applied to an analyte sample in the course of makingvoltammetric and/or amperometric measurements of analyte concentration.

The invention also provides a self-calibrating electrochemical analytesensor system which incorporates such an electrode. The invention alsoprovides a method of using such an electrode as an internalself-calibrating standard in an analyte sensor system, e.g. for thepurpose of making voltammetric and/or amperometric measurements todetermine the presence and/or concentration of an analyte in a sample.The invention also provides a method of making such an AIE.

The invention also provides a multi-phase AIE for use in anelectrochemical sensing device for measuring an analyte in a sample,comprising: (a) a first phase comprising an electrolytic layer (whichcan be, for example and without limitation, an ionic liquid (IL), whichin one embodiment is a liquid comprised solely of ions, wherein the ILphase is adjacent to the sample and substantially immiscible with thesample), (b) an electrically conductive component electrically connectedto the electrolytic layer, and (c) a redox active material (RAM),capable of being electrochemically oxidized and/or electrochemicallyreduced, wherein the redox activity of the redox active material issubstantially insensitive to the analyte, and wherein further the redoxactive material may be dispersed in either the electrolytic layer or theconductive component.

In some embodiments of the invention, the electrolytic layer is an IL,such as a room temperature IL (RTIL), i.e. a liquid comprised entirelyof ions which is liquid at temperatures below 100 degrees Celsius. Insome embodiments, the RTIL is N-butyl-N-methyl pyrrolidiniumbis(trifluoromethanesulfonyl)imide (C4mpyrrNTf2).

In various embodiments of the invention, the analyte is dispersed in aliquid sample, and/or is an ion dispersed in a liquid sample, and/or ishydrogen ion. In some embodiments, the analyte is a non-ionic speciesdispersed in a liquid species. In one aspect of the invention, the redoxactive material in the AIE is selected from the group consisting ofredox-active organic molecules, redox-active polymers, metal complexes,organometallic species, metals, metal salts, or semiconductors, andundergoes one or more electron transfer processes not involving anyreaction or chemical interaction with the target analyte. In someembodiments, the redox active material in the AIE is n-butyl-ferrocene.In other embodiments, the redox active material in the AIE is K₄Fe(CN)₆.

In some embodiments of the invention, the conductive component comprisesan electrically conductive material selected from the group consistingof carbon allotropes and derivatives thereof, transition metals andderivatives thereof, post-transition metals and derivatives thereof,conductive metal alloys and derivatives thereof, silicon and derivativesthereof, conductive polymeric compounds and derivatives thereof, andsemiconductor materials and derivatives thereof. In other embodiments ofthe invention, the conductive component further comprises a compositematerial comprising a binder and an electrically conductive material. Insome embodiments of the invention, the electrically conductive materialpresent in the composite material comprises graphite and/or glassycarbon, and/or multi-walled carbon nanotubes (MWCNTs) and/orsingle-walled carbon nanotubes (SWCNTs), and/or any combination thereof.In other embodiments of the invention, the composite material furthercomprises a redox active material. In one aspect, the redox activematerial is n-butyl-ferrocene. In other embodiments of the invention,the composite material comprises a redox-active ASM with conferredanalyte insensitivity as a consequence of the AIE construct.

In some embodiments of the invention, the AIE further comprises aconductive physical barrier adjacent to the sample, for physicallyseparating the electrolytic layer (e.g. IL phase) from the sample. Insome embodiments of the invention, the conductive physical barrier isselectively impermeable to the analyte. In some embodiments of theinvention, the conductive physical barrier is selectively permeable orselectively impermeable to non-analyte species in the sample. In otherembodiments of the invention, the conductive physical barrier is aporous frit. In some embodiments of the invention, the conductivephysical barrier is a membrane. In other embodiments of the invention,the conductive physical barrier is a film.

In some embodiments of the invention, the AIE further comprises a secondelectrolytic layer adjacent to the first electrolytic layer interposedbetween the conductive component and the first electrolytic layer and inelectrical connection with the conductive component and the firstelectrolytic layer, and a conductive physical barrier layer interposedbetween the first and second electrolytic layers, for physicallyseparating the electrolytic layers from each other, wherein, optionally,the first electrolytic layer is substantially immiscible with the secondelectrolytic layer and wherein further the redox active material mayalso be dispersed in the second electrolytic layer. In other embodimentsof the invention, the second electrolytic layer is selected from thegroup consisting of an aqueous electrolyte solution, a gelled aqueouselectrolyte solution, an electrolytic sol gel, and an organicelectrolyte solution.

The present invention further provides an electrochemical sensing devicefor measuring an analyte in a sample, comprising (a) an AIE as describedabove in any of its various embodiments, and (b) a working electrode inelectrical connection with the AIE, and comprising a redox-activeanalyte sensitive material (ASM), capable of being electrically oxidizedand/or electrically reduced, and wherein the redox activity of the ASMis substantially sensitive to the analyte. Working electrodes suitablefor use in the sensing devices of the invention include, for example andwithout limitation, those described in provisional U.S. patentapplication Ser. No. 61/161,139, filed 25 Mar. 09; Ser. No. 61/225,855,filed 15 Jul. 09; and Ser. No. 61/289,318, filed 22 Dec. 09, each ofwhich is incorporated herein by reference.

In some embodiments, the AIE and the working electrode are in parallelelectrical connection. In other embodiments, the AIE and the workingelectrode are configurationally joined and electrically connected by acommon conducting component. In another embodiment, the AIE and theworking electrode are electrically connected within a single-channelelectronic controlling device. In some embodiments, the AIE and theworking electrode are connected on separate data channels within amulti-channel electronic controlling device.

In some embodiments, the electrochemical sensing device of the presentinvention further comprises an electronic device for generating and/ormeasuring an analytical signal. In another embodiment, theelectrochemical sensing device of the present invention furthercomprises a conventional reference electrode or a pseudo-referenceelectrode.

The present invention further provides a method of using the sensingdevice of the present invention for measuring an analyte in a sample,comprising the steps of applying an electrical signal to the AIE, andmeasuring the electrical response of the AIE, wherein the electricalresponse of the AIE is independent of analyte concentration in thesample. In some embodiments, the measured electrical response of the AIEremains substantially constant over time and repeated use. In someembodiments, the measured electrical response of the AIE varies in asubstantially predictable manner over time and repeated use. In someembodiments, the measured electrical response of the AIE remainssubstantially constant relative to a reference potential, independent ofthe analyte, upon application of the electrical signal to the AIE. Inanother embodiment, the measured electrical response of the AIE variesin a substantially predictable manner relative to a reference potential,independent of the analyte, upon application of the electrical signal tothe AIE. In some embodiments, the measured electrical response of theAIE remains substantially constant relative to a known electricalresponse. In another embodiment, the measured electrical response of theAIE varies in a substantially predictable manner relative to a knownelectrical response.

These and other aspects and embodiments of the invention are describedin the accompanying drawings, which are briefly described below and inthe detailed description of the invention that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a conventional glass pH electrode which incorporates achloridized silver wire (Ag|AgCl) as the reference electrode.

FIG. 2 shows AIE configurations with which various analyte-insensitivematerials were tested, as shown in FIG. 3.

FIG. 3 shows a table presenting various AIM compounds that have beentested with various AIE configurations shown in FIG. 2. In the Figure,n-BuFc is n-butyl ferrocene; AG-np-CG is a silver nanoparticle-coatedglassy carbon; PVFc is polyvinyl ferrocene; and NiHCF is nickelhexacyanoferrate.

FIGS. 4A and 4B illustrate, in schematic form, exemplary embodiments ofthe invention. One or more redox active materials may be present ineither or both of the composite plug, or the electrolytic layer (e.g.the RTIL phase)

FIGS. 5A, 5B and 5C show results from voltammetric measurements takenfrom the third exemplary embodiment of the invention. FIG. 5A shows aplot of peak current vs. scan number measured over the course of 7000oxidative scans at pH 7 (1000 scans=430 minutes). FIG. 5B shows a plotof scan number vs. peak potential for the same experiment. FIG. 5C showsthe results from voltammetric measurements taken from a controlexperiment overlaid with the first 2000 scans from FIG. 5B. The controlexperiment is identical to the experiment used to generate the data inFIGS. 5A and 5B except that the electrolytic layers and conductivephysical barriers are absent from the construct.

FIGS. 6A, 6B, 6C and 6D show results of voltammetric measurements takenfrom the fifth exemplary embodiment of the present invention. FIG. 6Ashows pH buffer/KCl concentration vs. peak height measured over thecourse of 20 oxidative scans in several standard pH buffers as well as10 mM aqueous KCl solution and 100 mM aqueous KCl solution. FIG. 6Bshows a plot of pH buffer/KCl concentration vs. peak potential for thesame experiment. FIG. 6C shows the same data as FIG. 6B but rescaled onthe y-axis to allow a direct comparison to be made with the data in FIG.6D. FIG. 6D shows a plot of pH buffer/KCl concentration vs. peakpotential for the corresponding control experiment without the AIEconstruct (i.e. no RTIL in the construct). The peak potential scale forboth FIGS. 6C and 6D is the same.

FIG. 7 illustrates, in schematic form, exemplary embodiment 6 of theinvention without an intermediary layer and having a RAM in the plug.

DETAILED DESCRIPTION OF THE INVENTION

In the following description of the invention, specific conditionsrecited, for example, to prepare materials incorporated into embodimentsof the invention, may of course be varied in practice by those of skillin the art upon consideration of this disclosure. In general, theterminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “abinder” includes mixtures of binders, and a reference to “a conductivematerial” may include more than one such material. The followingparagraphs provide definitions for the convenience of the reader.

An “analyte” is a chemical species of interest present in a sample, thepresence of which is detectable or the concentration of which ismeasurable by using an analyte sensor system which incorporates the AIEof the present invention as a self-calibrating internal standard thatprovides either a substantially constant or a predictable responseduring analyte sensing. “Substantially constant” is used to meanconstant within a range defined by the end user.

A “redox active material” (RAM) is one that may be oxidized and/orreduced. “Redox activity” refers to either or both of those processes.

An “analyte insensitive material” (AIM), also known as a “chemicallyinsensitive redox active material”, is a redox active material that isinsensitive, or substantially insensitive, to the presence or theconcentration of an analyte in a sample. “Substantially insensitive” toan analyte is used to mean insensitive within the tolerances requiredfor a given application, as those tolerances are defined by an end user.Conversely, an “analyte sensitive material” (ASM) is a redox activematerial that (when not in an AIE of the invention) is sensitive orsubstantially sensitive to the presence or concentration of an analytein a sample within those user-defined application-specific tolerances.As discussed above, an ASM is functionally equivalent to an AIM whenutilized in an AIE of the invention. Thus, any reference to an AIM (orASM) should be considered to be a reference to an ASM (or AIM) inconnection with a description of a redox active material for use in anAIE of the invention. Thus, when a redox active material is part of anAIE of the invention, the term RAM may be used to indicate that anyredox active material (i.e. an AIE or an ASM) can be employed.

For a RAM to be “dispersed” means that it is dissolved in a solution orcolloidally suspended in a gas, liquid or solid. The term is alsointended to encompass embodiments wherein the RAM is abrasivelyimmobilized, adsorbed, electrostatically bound or covalently bound tothe surface of a solid or to a component of the solid. The term is alsointended to encompass embodiments wherein the RAM is incorporated as adopant in a crystal lattice. The term is also intended to encompass anintercalation of the RAM within a solid. In some embodiments of theinvention, the RAM is dispersed in a membrane that serves as aconductive physical barrier.

An “electrochemical sensing system” is a system of electrodes which iscapable of measuring the presence and/or concentration of an analyte ina sample. Such systems generally include a working electrode, areference electrode (either a conventional or pseudo referenceelectrode) and a counter electrode. Optionally, e.g., in cases where theworking electrode is a microelectrode, the system may include only areference electrode and working electrode. Optionally, the system mayinclude a controller/processor device.

A “working electrode” (WE) is the electrode at which the electrochemicalprocess of interest occurs. In a sensor, the working electrode may besensitive to one or more analyte(s) in the test solution, or it may bechemically modified with analyte sensitive species/materials. Theelectrochemical response of the working electrode is measured after someperturbation has been applied to the system under study. For example,the perturbation may be the application of a potential difference to theWE which induces electron transfer to occur, and the resulting currentat the working electrode is then recorded as a function of either theapplied potential (voltammetric mode) or time (chronoamperometric mode).These two examples of modes of operation are illustrative and notexhaustive; many other modes are known in the art.

An “analyte insensitive electrode” (AIE) is a special case of a workingelectrode where the current flow depends in part on redox processes thatare independent of the presence or concentration of species (apart froma minimum threshold of supporting electrolyte) in the sample compositionincluding but not limited to the analyte. AIEs of the invention aredescribed in more detail below.

To monitor the potential difference applied to the WE, a reference pointis required. This is provided by the use of a “reference electrode”(RE). Conventional reference electrodes (CREs) have a certain fixedchemical composition and therefore a fixed electrochemical potential,thus allowing measurement of the potential difference applied to the WEin a known, controlled manner. A CRE typically comprises two halves of aredox couple in contact with an electrolyte of fixed ionic compositionand ionic strength. Because both halves of the redox couple are presentand the composition of all the species involved is fixed, the system ismaintained at equilibrium, and the potential drop (i.e. the measuredvoltage) across the electrode-electrolyte interface of the CRE is thenthermodynamically fixed and constant. For example a commonly used CREsystem is the Ag|AgCl|KCl system with a defined and constantconcentration of KCl. The two half-cell reactions are therefore:Ag⁺+e⁻→Ag; and AgCl+e⁻→Ag+Cl⁻. The overall cell reaction is therefore:AgCl→Ag⁺+Cl⁻ for which the Nernst equilibrium potential is given as:E=E⁰−(RT/F)*ln[Cl⁻] where E is the measured RE potential, E⁰ is thestandard potential of the Ag|AgCl couple vs. the standard hydrogenelectrode with all species at unit activity (by convention this isdefined as having a potential of 0.0V), R, T and F are the universal gasconstant, temperature and Faraday constant respectively in appropriateunits. Hence the potential of this system depends only on theconcentration (more strictly speaking the activity) of CF ion present,which, if this is fixed, provides a stable, fixed potential. Many otherCRE systems are known in the art. It is imperative that the compositionof the CRE remains constant, and hence almost no current should bepassed through the CRE (otherwise electrolysis will occur and thecomposition of the CRE will change), which necessitates the use of athird electrode, the counter electrode (CE) to complete the circuit.However, two-electrode configurations can be used in the special casewhere the WE is a microelectrode, having at least one dimensiontypically smaller than 100 microns. In this case, the currents passed atthe WE are small, and therefore a two-electrode cell can be used with aCRE, but without the need for a CE.

The term “pseudo-reference electrode” (PRE) refers to a type ofreference electrode which is sometimes used, particularly in non-aqueouselectrolytes. These electrodes typically do not comprise both halves ofa well-defined redox potential and are therefore not thermodynamicreference electrodes of fixed composition and potential. However, theyprovide a reasonably constant potential over the timescale of anelectrochemical experiment (on the order of minutes), and the absolutepotential of the PRE can then be calibrated back to a CRE if required.One example of a PRE is a silver wire (used commonly in non-aqueouselectrochemistry).

To pass current through the cell, one further electrode is required tocomplete the circuit, known as a “counter electrode” (CE) or sometimesan “auxiliary electrode”. This electrode simply serves as a source orsink of electrons and allows current to flow through the cell. To avoidunwanted electrochemical redox processes occurring at the CE, which mayinterfere with the signal measured at the WE, CEs are typically madeusing relatively chemically inert materials, commonly Pt, but carbon(graphite) is also commonly employed.

A “conductive physical barrier” is a layer that is either adjacent tothe sample being analyzed or is interposed between two adjacent phasesof the AIE. A conductive physical barrier is “selectively impermeable”to a species in a sample when it prevents the species from passingthrough it but allows other components of the sample, such as chargecarriers in the electrolyte component of the sample, to do so freely.Conversely, a conductive physical barrier is “selectively permeable” toa species in the sample when it allows the species to move freely acrossit. In some embodiments, the AIE may incorporate more than oneconductive physical barrier as a means of physically separating thevarious components of the AIE from one another as well as from thesample being analyzed.

“Configurationally joined” denotes a direct physical connection betweentwo or among several elements.

An “ionic liquid” (IL) is a liquid comprised principally of both cationsand anions. In one embodiment, an “ionic liquid” (IL) is a liquidcomprised entirely of both cations and anions. A “room temperature ionicliquid” (RTIL) is an IL that is a liquid at temperatures below 100degrees Celsius.

Phases which are “adjacent” to another phase, i.e., to the conductivecomponent or to the sample, may optionally be physically separated by aninterface layer.

With these definitions in mind, one of ordinary skill in the art canbetter understand the organization and content of the followingdiscussion of the invention. A description of the Analyte InsensitiveElectrode (AIE) of the invention and its uses is followed by discussionof the individual components of the AIE, culminating in severalexemplary embodiments of the AIE. Upon conclusion of the description ofthe AIE itself, an electrochemical sensing system that uses an AIE ofthe invention is then described, including a description of the variouscomponents of said system.

Prior to the present invention, typical voltammetric or amperometricelectrochemical sensing systems typically comprised three electrodes,the working electrode, the reference electrode, and the counterelectrode. Under conditions where little current flows through thesensing system, the reference and counter electrode functions can becombined into a single electrode resulting in a two-electrode system(working and reference/counter). Briefly, the sensing system functionsby monitoring the current flow through the WE as a function of theapplied potential. The applied potential is monitored by the RE, whilethe potential and current are supplied to the sample by the CE. Thepotential sensed by the RE is continuously compared with the desiredwaveform output by the controller/processor device. If the potentialrecorded by the RE does not reflect the desired potential, then thecounter electrode is adjusted until the RE potential and the desiredpotential match. The accuracy of the current versus voltage response ofthe system therefore depends on the ability of the RE to measure thepotential at the WE accurately. High accuracy thus necessitates a highlystable RE that effectively measures the potential without being affectedby the sample or duration of measurement. The silver/silver chloride andsaturated calomel electrodes are examples of such conventional referenceelectrodes (CREs).

As discussed above, CREs such as silver/silver chloride and saturatedcalomel electrodes have several disadvantages. There remains a need fora system that circumvents these disadvantages. The present inventionaddresses this by loosening the stability requirement of the RE viaintroduction of a fourth electrode, the AIE. Due to this relaxation instability, simple pseudo reference electrodes (PREs), such as the silverwire, may be used as REs. Functionally the AIE behaves as an additionalWE with the exception that the AIE response, being independent ofanalyte, is always fixed relative to a known response such as thestandard hydrogen electrode (SHE). Thus, instability in a given RE canbe corrected by comparison with the known fixed response of the AIE.

In comparison to examples from the prior art that include pH insensitiveredox materials or other AIMs (see for instance WO2005/066618 orWO2005/085825), the utility of the AIE of the invention arises, at leastin part, from its ability to improve the stability and longevity of theAIM across both time and analyte composition. These improvements act toincrease the performance and scope of applicability of the AIM comparedto the prior art. Furthermore, the AIE construct relaxes the requirementthat the RAM employed be insensitive to pH or other analytes ofinterest, thus broadening the range of RAMs that may be used to includenot only AIMs but ASMs as well.

Thus, in a first aspect, the present invention provides an electrodethat renders redox materials contained therein insensitive tonon-electrical external influences (e.g. analyte concentrations) whilemaintaining electrical contact with the balance of the components of thesensing system. The electrode of the invention is therefore an analyteinsensitive electrode (AIE). Due to this analyte insensitivity, thesignal response of the AIE is independent of the environment in whichthe sensing system is placed, and therefore can be used as an internalcalibration point. The AIE can comprise a redox active material that isitself insensitive to the analyte (i.e. an analyte insensitive materialor AIM) independent from the construct. Due to the nature of theconstruct, however, it is not a requirement that the redox material beinsensitive to the analyte independent of the construct. In this case,for example, the AIE may comprise a redox material which is itselfsensitive to the analyte (i.e. an analyte sensitive material or ASM)independent from the construct, but which is rendered insensitive to theanalyte when used as the redox component of the AIE.

Analyte Insensitive Electrode (AIE)

An AIE of the present invention can be viewed as a special class of WEin that it operates in much the same way as the WE described above,except that the measured AIE response is substantially insensitive tochanges in the composition and/or presence of analyte(s) within the testsolution. It does, however, comprise a redox-active species and soprovides an electrochemical response, but one which is independent ofany changes to the test solution composition (apart from a minimumthreshold of supporting electrolyte) or the presence of any analyte.Thus, the AIE provides a constant, fixed electrochemical response, thenature of which depends on the mode of operation. For example, in avoltammetric mode of operation, the AIE of the invention can produce asignal which has a constant peak current or alternatively a constantpeak potential relative to the standard hydrogen electrode (SHE) or itmay have both fixed peak current and fixed peak potential (relative tothe SHE). This signal can then be used as an internal standard fromwhich the signal at the WE produced due to the presence of some targetanalyte(s) may be calibrated. Because this signal is constant relativeto a known fixed quantity, such as the SHE, it may be used in practicewith a less stable reference electrode such as a PRE thus providing astable signal for internal calibration without the use of a conventionalreference electrode.

In one aspect, the invention provides a multi-phase AIE for use in avoltammetric and/or amperometric electrochemical sensor system formeasuring the presence and/or concentration of an analyte in a sample.The AIE provides a predictable analyte-insensitive signal which is usedas a standard of comparison for the signal generated using an ASM at theworking electrode, and thereby allows an end user to determine ananalyte's presence and/or concentration. An advantage of embodiments ofthis aspect of the invention is that it provides a self-calibrationmeans, and thereby obviates any need for repeated calibration by the enduser.

The AIE provided by the invention can include the following components:an electrolytic layer, a redox active material (RAM), a conductivecomponent, and, optionally, a conductive physical barrier. In someembodiments, one or more of these components may be combined together.For instance, the RAM may be dispersed in the electrolytic layer and/orthe electrolytic layer may saturate the pores of the conductive physicalbarrier. In other embodiments, the AIE optionally includes more than oneof any one or more of the components described above.

Conductive Physical Barrier

The conductive physical barrier, when present, serves to separatephysically the other components of the AIE from the analyte. Thisphysical separation attenuates the direct chemical interaction of theremainder of the AIE with the analyte, thereby minimizing effects suchas convective mixing between the analyte and electrolytic layer. This inturn minimizes the change in composition of the electrolytic layer dueto interaction with the analyte. A requirement of the conductivephysical barrier is that it effectively conducts the current necessaryfor generating the electrical signal associated with the redox activematerial.

Some embodiments of the invention have a single conductive physicalbarrier between the electrolytic layer and the analyte, while otherembodiments include additional conductive physical barriers that serveto separate physically but maintain electrical contact between multipleelectrolytic layers within the AIE. In other embodiments of theinvention, a conductive physical barrier is present at the open end ofthe AIE housing, interposed between the sample and the contents of theAIE cavity, to contain the contents within the AIE cavity. In anotherembodiment, one or more internal conductive physical barriers arepresent within the AIE cavity, at the interface between any two of thecomponent phases within the AIE cavity, to separate those two phasesfrom each other physically.

Selection criteria for the conductive physical barrier include but arenot limited to tensile strength, wettability and porosity. Suitablematerials include, but are not limited to, membranes, porous frits, andfilms. In some embodiments, the conductive physical barrier comprises aRAM. For example, the RAM may be dispersed in a conductive physicalbarrier. In some embodiments, a conductive physical barrier is amembrane containing an IL containing the RAM. In other embodiments, theconductive barrier is a polyethersulfone membrane. In anotherembodiment, the conductive physical barrier is a PVDF (polyvinylidenedifluoride) membrane. Additional embodiments may combine the conductivephysical barrier with the electrolytic layer into one component.

Electrolytic Layer

The electrolytic layer (e.g. composed of an RTIL or other suitablematerial, as described herein) provides the constant chemicalenvironment and ionic strength for the RAM and provides a layer thatlimits or eliminates direct chemical interaction of the RAM with thesample being analyzed. Selection criteria for the electrolytic layerinclude (a) that its component composition must remain substantiallyunchanged over the lifetime of the AIE, (b) that it effectively conductsthe current necessary for generating the electrical signal associatedwith the redox active material, and, optionally, (c) that it besubstantially immiscible with the sample being analyzed.

In some embodiments, the electrolytic layer is comprised of a fluorouslayer with a dispersed electrolyte. Suitable fluorous organic liquidsinclude but are not limited to perfluoroaromatic compounds (e.g.hexafluorobenzene), perfluoroalkanes (e.g. tetradecafluorohexane,octadecafluorooctane, eicosafluorononane, and decafluoropentane) andalkyl perfluoroalkyl ethers (e.g. nonafluorobutyl methyl ether). Thedispersed electrolyte for the fluorous layer can be but is not limitedto a fluorous ionic liquid (for examples see: Chem. Comm. 2000,2051-2052, incorporated herein by reference).

In some embodiments, the electrolytic layer is a fluorous phasecomprising a fluorous organic liquid and a dispersed electrolyte that isadjacent to but substantially immiscible with the sample. In variousembodiments, the fluorous phase is at least 50% or at least 90% byweight of one or more fluorous organic compounds and the balance of thephase is comprised of one or more diluents including but not limited togelling agents, electrolytes, organic solvents, water, inorganiccompounds including salts, organic compounds, carbon allotropes, andredox active materials. In some embodiments of the invention, more thanone fluorous phase may be present.

In some embodiments, the electrolytic layer is comprised of an organiclayer with a dispersed electrolyte. For example, a phase transfer agentdissolved in a common organic solvent (e.g. tetrabutyl ammonium bromidein toluene) is a suitable electrolytic layer.

In some embodiments, the electrolytic layer is an organic phasecomprising an organic liquid adjacent to but substantially immisciblewith the sample. In various embodiments, the composition of the organicphase is at least 50% or at least 90% by weight of one or more organiccompounds and the balance of the phase is comprised of one or morediluents including but not limited to gelling agents, electrolytes,water, inorganic compounds including salts, organic compounds, carbonallotropes, and redox active materials. In some embodiments of theinvention, more than one organic phase may be present.

In some embodiments, the electrolytic layer is comprised of an aqueouslayer with a dispersed electrolyte. The aqueous layer is comprisedsubstantially of water but may also include one or more diluentsincluding but not limited to gelling agents, organic solvents, inorganiccompounds including salts, organic compounds, carbon allotropes, andredox active materials. The dispersed electrolyte comprises a salt ofanions and cations chosen from a group including but not limited toinorganic, organic, and/or polymeric ions. In one embodiment thedispersed electrolyte is potassium chloride.

Due to the inherent low volatility of ionic liquids (ILs) and theirionic, and therefore, electrically conductive nature, ILs meet thecriteria described above for electrolytic layers and so are employed invarious embodiments of the invention. In some embodiments of the presentinvention, the electrolytic layer phase generally includes at least oneIL that is substantially immiscible with the sample or with anyintermediary electrolytic layers or phases, when such phases arepresent. This quality of substantial immiscibility may be achieved as aresult of the intrinsic properties of the IL itself, or may be theresult of components such as a conductive physical barrier present inthe construct (e.g. a porous frit) separating the phases or a membraneseparating the phases. The particular components of the IL phase may bechosen to achieve desired characteristics, which may include but are notlimited to sample immiscibility; temperature stability; viscosity,dielectric constant; specific ionic chemical composition, and phasestate at temperatures characteristic of particular applications.

In some embodiments, the IL components are chosen so as to be liquid ata temperature or within a range of temperatures that include thetemperature within which the sample will be measured. Suitabletemperature ranges include but are not limited to between 10 degrees to50 degrees Celsius; alternately from about 16 degrees to about 45degrees Celsius; alternately from about 20 degrees to about 40 degreesCelsius.

In one embodiment, the electrolytic layer is an ionic liquid (IL)composed of an anionic chemical species and a cationic chemical speciesand located adjacent to the sample but substantially immiscible with thesample. In various embodiments, the composition of the IL phase is atleast 50% or at least 90% by weight of one or more ionic liquids and thebalance of the phase is comprised of one or more diluents including butnot limited to gelling agents, electrolytes, organic solvents, water,inorganic compounds including salts, organic compounds, carbonallotropes, and redox active materials, In some embodiments, thecomposition of the IL phase is 100% of one or more ionic liquids.Illustrative IL cations include but are not limited to quaternarypyrrolidines and N,N′-disubstituted imidazoles. Illustrative IL anionsinclude but are not limited to imides and borates, phosphates, andsulphates, which may be substituted as appropriate to form an anion ofinterest.

In various embodiments, the IL cation is selected from a group thatincludes but is not limited to imidazolium (for example,1-butyl-3-methylimidazolium, C₄mim), pyridinium (for example, N-butylpyridinium (C₄py)), pyrrolidinium (for example, N-butyl-N-methylpyrrolidinium (C₄mpyrr)), tetraalkylammonium, and tetraalkylphosphonium.In various aspects, the IL anion is selected from a group that includesbut is not limited to tetrafluoroborate (BF₄),bis(trifluoromethanesulfonyl)imide (N(Tf)₂), thiocyanate (SCN),dicyanamide (N(CN)₂), ethyl sulphate ((EtSO₄), hexafluorophosphate,(PF₆) and trifluorotris(pentafluoroethyl)phosphate (FAP). In oneembodiment, the IL is an RTIL. In some embodiments, the RTIL phase isN-butyl-N-methyl pyrrolidinium bis(trifluoromethanesulfonyl)imide([C₄mpyrr][N(Tf)₂]).

In some embodiments, the electrolytic layer comprises a microporousmaterial wherein the IL or RTIL is immobilized within its interconnectedmicropores. For example, silica based sol-gels can be formed in thepresence of ILs resulting in an ionogel which is conductive as a resultof the IL contained therein (for example see: Chem. Mater., 2006, 18(17), pp 3931-3936, incorporated herein by reference).

Alternate microporous materials suitable for use in the AIEs of theinvention include those well known in the field of synthetic membranes.Membranes used for selective separations cover a broad range of poresizes ranging from micrometers to nanometers, and may be derived fromorganic materials, especially polymers. For example, microporousmembranes based on polysulfone, polyethersulfone,polyvinylidenefluoride, polytetrafluoroethylene, and certain derivativesbased on those polymers generally exhibit good chemical stability towardboth typical analyte solutions and the RTIL, and are thus suitable mediafor immobilizing the RTIL In this case the RTIL is the principal conduitof charge transfer across the electrolytic layer.

Yet another embodiment of the electrolytic layer is a thin layer ofnonporous solid material, for example a polymer film, in which the RTILexhibits some solubility. The polymer film containing dissolved RTIL canbe considered as a solid solution, in that it exhibits the ionicconducting characteristic of the RTIL, but retains the dimensionalstability of a solid. Compared to a microporous structure, which musthave a minimum thickness to retain a liquid effectively, a nonporoussolid solution film can be made extremely thin without developingdefects or pinholes. Examples of suitable nonporous polymer filmsinclude, but are not limited to, cross-linked derivatives ofpolyimidazole, poly(vinyl alcohol), poly(vinyl acetate), poly(ethyleneoxide), and their copolymers or blends. Conductivity in the solidsolution film may be established at different loading, or concentration,of RTIL in different polymers. Non-cross-linked polymers may be usedwhere a relatively low loading of RTIL is sufficient to obtainconductivity. Cross-linked polymers are preferred at relatively highloadings of RTIL to prevent excessive swelling or dissolution. In thiscase the solid solution of RTIL is a substantially uniform conduit ofcharges. The solid solution film can be surface-modified to manipulateits selectivity toward hydrogen ions.

The AIE optionally includes an additional electrolytic layer. In oneembodiment, an electrolytic layer is adjacent to the sample, and anadditional electrolytic layer is interposed between any of the otherphases of the electrode. Optionally, two electrolytic layers may beadjacent to each other. Selection criteria for the additionalelectrolytic layer include the criteria described above for electrolyticlayers. Selection criteria for an additional electrolytic layercomposition include but are not limited to viscosity and dielectricconstant. Exemplary materials include but are not limited to materialsas described above for the first electrolytic layer, an ionic liquid, anaqueous electrolyte solution, a gelled aqueous electrolyte solution, anelectrolyte containing sol-gel, an electrically conductive sol-gel, anorganic solvent, and an organic electrolyte solution.

Optionally, an electrolytic layer comprises one or more RAMs. RAMssuitable for use in an electrolytic layer include, but are not limitedto, the RAMs described below.

Redox Active Materials (RAMs)

Selection criteria for suitable RAMs that are a component of the AIE ofthe present invention include, but are not limited to, oxidation and/orreduction peaks obtained during voltammetric and/or amperometricmeasurements which are well-defined and are either substantiallyconstant or vary in a definable manner. As discussed above, either anAIM or an ASM can be used in the AIE of the invention. In the AIE, anASM is effectively transformed into an AIM due to being isolated fromthe sample by the electrolytic layer. For example, AQ and PAQ are ASMs,but if they are isolated from any test solution either by beingdispersed in the electrolytic layer or in contact with an aqueous phaseof fixed pH behind the electrolytic layer, and the electrolytic layer(e.g the RTIL) prevents the transfer of protons, then there is one fixedsignal (the non-aqueous voltammetry of AQ or PAQ in the electrolyticlayer) or the usual (but constant) signal in the aqueous layer. Hencethe ASM would then be considered an AIM within the context of thisembodiment of the invention. The AIE construct thus confers analyteinsensitivity to an ASM.

Suitable redox active materials for use in the AIE of the presentinvention are reversible, quasi-reversible and irreversible redox-activecompounds, including but not limited to redox-active organic molecules,redox-active polymers, metal complexes, organometallic species, metals,metal salts or semiconductors, present as liquids or solids formed asbulk materials, microparticles or nanoparticles, that undergo one ormore electron transfer processes not involving reaction with the targetanalyte and whose redox behaviors are therefore insensitive to thepresence of the target analyte. As noted above, an ASM can also be usedto form the AIE of the invention if it is isolated from the sample bythe electrolytic layer.

In various embodiments of the invention, the RAM is selected fromcompounds that include but are not limited to, ferrocene or ferrocenederivatives including but not limited to ferrocene derivativescomprising alkyl, aryl and heteroatomic substituents on one or bothcyclopentadienyl rings; polymers of variable cross-linking comprisingferrocene and other non-redox-active monomers such as styrene andacrylates, silver nanoparticles on carbon substrates such as glassycarbon, graphite, and carbon nanotubes (CNTs), hexacyano iron compoundswith variable counter-ions including but not limited to main group andtransition metal cations, and other redox-active transition metalcomplexes.

In various embodiments, the RAM is n-butyl-ferrocene, silvernanoparticle modified glassy carbon powder (AG-np-GC), ferrocene,polyvinylferrocene, nickelhexacyanoferrate, ferrocene styrenecopolymers, ferrocene styrene cross-linked copolymers, Ni Cyclam, orK₄Fe(CN)₆. In some embodiments, the AIM is n-butyl-ferrocene. In anotherembodiment, the AIM is K₄Fe(CN)₆. This list is not exhaustive and is notintended to be limiting. One skilled in the art will be able to identifyand use many other RAMs in place of or in addition to those listedabove.

As discussed above, an ASM can serve the same function as an AIM in theAIE construct. Suitable ASMs for use in the AIE include but are notlimited to anthraquinone (AQ), anthracene, 9,10-phenanthrenequinone(PAQ), 1,4-benzoquinone, 1,2-benzoquinone, 1,4-napthaquinone,1,2-napthaquinone, N,N′-diphenyl-p-phenylenediamine (DPPD), azocontaining compounds such as azobenzene and derivatives thereof,porphyrins and derivatives thereof such as octaethylporphyrin ortetraphenylporphyrins, metalloporphyrins and derivatives thereof such ashemin, iron octaethylprophyrin, iron tetraphenylporphyrin, and viologensand derivatives thereof such as methyl viologen. This list is notexhaustive and is not intended to be limiting. One skilled in the artwill be able to identify and use many other ASMs in place of or inaddition to those listed above.

One or more RAMs may be dispersed in one or more of any of theelectrolytic layers (e.g the IL) or the external conductive physicalbarrier or the conductive component, or may be dispersed in an internalconductive physical barrier. In other embodiments, a single RAM isdispersed in one or more of the electrolytic layers (e.g. the IL) or inthe conductive component. In another embodiment, different RAMs aredispersed in one or more electrolytic layers or in the conductivecomponent.

Conductive Component

The AIE of the current invention includes a conductive component. Insome embodiments, the conductive component is a conductive backinglocated at the rear of the AIE cavity and forming the rear wall of thecavity. A conductive lead (sometimes referred to herein as atransmission element) is electrically connected to the backing (i.e. bybeing soldered to the backing), and protrudes through the rear bore,which may optionally be sealed with one or more materials including butnot limited to a silicone polymer, including a substituted siliconepolymer and an epoxy composition. The lead provides a means oftransmitting electrical signals to and from the electrode.

A variety of conductive materials may be used for the conductivebacking, including but not limited to carbon allotropes and derivativesthereof, transition metals (e.g platinum, gold, and mercury), conductivemetal alloys, conductive polymeric compounds and derivatives thereof,semiconductor materials and derivatives thereof, including silicon andderivatives thereof, and additional suitable materials not specificallymentioned.

Optionally, a RAM is associated with the conductive backing by methodsincluding but not limited to abrasive immobilization, adsorption,electrostatic binding or covalent binding to the backing surface.Suitable RAMs for associating with the conductive backing include butare not limited to those specified in the preceding section.

Optionally, the conductive component may comprise a plug of conductivematerial in addition to or in place of the conductive backing. In thelatter case, the transmission element is electrically connected to theplug. In one aspect, the plug contains a binder and at least oneelectrically conductive material. Suitable binders include but are notlimited to epoxy, mineral oil and polymer binders. Suitable conductivematerials include but are not limited to graphite, MWCNTs, SWCNTs,glassy carbon, as well as those discussed above as materials useful forthe conductive backing.

In other embodiments the plug is a mixture of epoxy and graphite. Inanother embodiment, the plug is a mixture of epoxy, graphite and MWCNTs.In another embodiment, the plug is a mixture of epoxy, graphite andSWCNTs. In another embodiment, the plug is a mixture of epoxy, graphiteand glassy carbon. In another embodiment, the plug is formed using epoxyand MWCNTs. In another embodiment, the plug is formed using epoxy andSWCNTs. In another embodiment, the plug is formed using epoxy and glassycarbon. Optionally, one or more RAMs are present in the composite plug,in addition to the binder and the conductive material. RAMs suitable forassociating with or incorporating into the conductive plug include butare not limited to those specified in the preceding section. In someembodiments, the RAM is mixed with the binder and the conductivematerial. In another aspect, the RAM is associated with one of thecomponents of the composite material, by methods including but notlimited to abrasive immobilization, adsorption, electrostatic binding orcovalent binding. In other embodiments, the components are combined viamechanical mixing using a mortar and pestle. In another embodiment thecomponents are mixed in an organic solvent.

Exemplary Configurations of the AIE

A number of configurations of the component elements of the claimed AIEare possible. Exemplary embodiments are shown schematically in FIGS. 4Aand 4B. As shown in FIGS. 4A and 4B with labeled parts, aspects of theAIE include a cylindrical housing (1) with the conductive backing (5) ofthe conductive component (4) defining an AIE cavity (3) having a sampleend (12). A conductive lead (2) is attached and in electrical connectionwith the conductive plate and protrudes through the rear bore, providinga means for transmitting electrical signals to and from the AIE. Theconductive component optionally includes a plug of conductive compositematerial (6) in the AIE cavity in electrical contact with and adjacentto the conductive backing. A first electrolytic layer (e.g IL phase) (7)is adjacent to the conductive component, and contained within the AIEcavity by a first, external, conductive physical barrier (8).Optionally, additional electrolytic layers (e.g. IL phases) may bepresent. Optionally, an additional electrolytic layer (10) is locatedbetween the conductive component and the first electrolytic layer (e.g.IL phase), and is separated from the first electrolytic layer (e.g. ILphase) by a conductive physical barrier (9). Optionally, additionalelectrolytic layers may be present.

The RAM may be dispersed in the conductive component, optionally ineither the conductive backing and/or the composite plug, or in any ofthe electrolytic layers (e.g. IL layers). In operation, the analyte ischemically isolated from the RAM by the electrolytic layer (e.g. ILlayer). The RAM may be further chemically isolated or physicallyisolated from the analyte by an additional electrolytic layer.

Some embodiments of the invention are illustrated in FIG. 4A, asdescribed above. The conductive component (4) includes a conductivecomposite plug (6) and an electrolytic layer (e.g. an IL phase) (7),contained by an external conductive physical barrier (8). There may beRAM dispersed in the plug or in the electrolytic layer (e.g. IL phase).Optionally, the same or different RAMs are dispersed within both theplug and the electrolytic layer (e.g. IL phase).

In another embodiment of the invention (FIG. 4B), a second electrolyticlayer (10) is added between the conductive material plug and the firstelectrolytic layer (e.g. IL phase). The second electrolytic layer may bein direct contact with the first electrolytic layer (e.g. IL phase);alternatively, a first internal conductive physical barrier (9) mayseparate the second electrolytic layer from the first electrolytic layer(e.g. IL phase). One or more RAMs may be dispersed in the conductivecomponent or the electrolytic layers (e.g. IL phase).

In one embodiment, the present invention provides a multi-phase AIE foruse in an electrochemical sensing device for measuring an analyte in asample, the AIE comprising (a) an electrolytic layer; (b) anelectrically conductive component electrically connected to theelectrolytic layer, and (c) a redox active material (RAM), capable ofbeing electrically oxidized and/or electrically reduced, wherein theredox activity of the material is substantially insensitive to theanalyte, and wherein further the RAM may be dispersed in either theelectrolytic layer or the conductive component.

Electrochemical Sensing System

In another aspect, the present invention provides an electrochemicalsensor system for measuring the presence and/or concentration of ananalyte in a sample. Various embodiments of the invention are possible,which have in common an embodiment of the AIE of the present invention,a working electrode, optionally a counter electrode, a reference orpseudo-reference electrode and a controller device for supplying a rangeof electrical signals to the indicator and working electrodes, andmeasuring the electrical response of the working electrode and the AIEover the range of applied signals. These components are discussed infurther detail below.

System Component: Analyte Insensitive Electrode

The analyte sensor system of the present invention includes an AIE asdescribed above.

System Component: Working Electrode

The analyte sensing system of the present invention further includes aworking electrode. Working electrodes suitable for use in the sensorsystem of the present invention are known in the art. See U.S. Pat. No.5,223,117, PCT Patent Publication Nos. 2005/066618 and 2007/034131 andGB Patent Publication No. 2409902. Working electrodes suitable for usein the sensing devices of the invention include, for example and withoutlimitation, those described in provisional U.S. patent application Ser.No. 61/161,139, filed 25 Mar. 09; Ser. No. 61/225,855, filed 15 Jul. 09;and Ser. No. 61/289,318, filed 22 Dec. 09, each of which is incorporatedherein by reference.

Characteristic of the working electrode component of the presentinvention is that it allows the passage of current, in response toelectrical perturbations of the sample, and demonstrates anelectrochemical response that is sensitive to one or more analyte(s) inthe system. Optionally, the WE may be chemically modified with analytesensitive species or materials. In one aspect, the WE is modified withat least one analyte-sensitive redox active material having well-definedoxidation and/or reduction peaks.

The general mode of operation of working electrodes in anelectrochemical sensor system is known in the art. Upon being subjectedto an electrical signal (optionally, an applied potential relative tosome thermodynamically fixed reference electrode potential), theelectrical response of the working electrode is measured and compared toa reference point provided by, for example, an external calibration plotin cases where the WE passes minimal current (potentiometric device),or, in embodiments of the present invention, to a reference pointprovided by the AIE. In voltammetric mode, the WE response is measuredas a function of the potential difference applied between the WE andsome suitable CRE/PRE.

System Component: Reference Electrode

The sensor system of the present invention includes a conventionalreference electrode or, a pseudo-reference electrode. Examples ofconventional reference electrodes and pseudo-reference electrodes areknown in the art. See Bard and Faulkner, “Electrochemical Methods:Fundamentals and Applications” (Wiley 2001). In operation, the CREallows the application of a known, controlled potential difference tothe WE by providing a fixed reference point.

In some embodiments, a “pseudo-reference electrode” (PRE) may be used.These typically do not comprise both halves of a well-defined redoxpotential, and are therefore not thermodynamic reference electrodes offixed composition and potential. However, they are functionally simplerthan a conventional RE and provide a reasonably constant potential overthe timescale of an electrochemical experiment. Use of the PRE inconjunction with an AIE of the present invention obviates the need forthe conventional reference electrode thereby overcoming thedisadvantages of the CRE. One (but not exhaustive) example of a PRE isthe use of a silver wire.

System Component: Counter Electrode

Counter electrodes suitable for use in the sensor system of the presentinvention are known in the art. See, for example, Bard and Faulkner,“Electrochemical Methods: Fundamentals and Applications” (Wiley 2001).Optionally, in order to avoid unwanted electrochemical redox processesoccurring at the CE which may interfere with the signal measured at theWE, the CE is typically made of a relatively chemically inert material,commonly Pt or carbon (graphite). In operation, the CE serves as anelectron source or sink, thereby delivering current to the sample andallowing it to flow through the sensor system.

System Component: Controller/Processor Device

The analyte sensor system of the present invention further includes acontroller/processor device. Controller/processor devices suitable foruse in the analyte sensor system of the invention include, for exampleand without limitation, those described in provisional U.S. patentapplication Ser. No. 61/161,139, filed 25 Mar. 2009; Ser. No.61/225,855, filed 15 Jul. 2009; Ser. No. 61/289,318, filed 22 Dec. 2009;Ser. No. 61/308,244, filed 25 Feb. 2010; and Ser. No. 61/309,182, filed1 Mar. 2010. In other embodiments, the controller/processor device is asingle-channel device through which the working electrode and AIE areelectrically connected and controlled, and their signals recorded, onthe same channel. Examples of single channel controllers include apotentiostat and a galvanostat. In another embodiment of the invention,the working electrode and the AIE are physically remote and connected bya multi-channel device capable of controlling and/or recording signalsfrom the working electrode and the AIE independently. In someembodiments, the controller/processor device is a multichannelpotentiostat. In yet another embodiment, the signals from the workingelectrode and AIE are combined and the processor then analyzes the datafrom the combined signal. In another embodiment, the signals from theworking electrode and AIE are recorded separately and subsequentlycombined and analyzed by the processor.

Voltammetric Measurement

The electrical responses of the AIE as incorporated in embodiments ofthe sensor system of the present invention are determined using methodsincluding but not limited to cyclic or square-wave voltammetry asdescribed in the “Materials and Methods” section of the Example below.In operation, upon being subjected to cyclic or square wave voltammetry,embodiments of the present invention give an electrical response that issubstantially constant or varies in a substantially predictable manner.

While one important application of the present invention is themeasurement of pH, the invention has application in all areas involvingvoltammetric and amperometric sensing, including but not limited to thedetection of metal ions, metal complexes and derivatives (e.g. As(III),Pb(II), Fe(II/III), Cu(II/I), Hg(II), and many others), the detection ofpollutants (e.g. chlorinated phenols/organics, pesticides, herbicides,nitrite/nitrate, and the like), gas sensing both directly in air anddissolved gases in aqueous media (e.g. CO₂, CO, SO₂ and H₂S inparticular—important in the petrochemical and automobile industries,among many others), environmental monitoring (WHO, US EPA, and EUregulatory bodies), drug detection (e.g. the OxTox drug testing unit),food industry Q&A (e.g. capsaicin in spicy foods, hesperidin in citrusfruit juices, and the like), medical and (bio)pharmaceutical Q&A,diagnostics/accreditation, and glucose sensing (diabetes, homemonitoring). Accordingly, those of skill in the art will recognize thatthe devices and methods of the present invention are illustrated by butare not limited by the examples that follow.

EXAMPLES Materials and Methods: Conductive Components Graphite/EpoxyComposite

A graphite/epoxy composite was prepared by combining 300 mg graphitewith 1.15 grams of epoxy A and 150 mg of epoxy B (Epoxy Technology,Bellerica, Md.) using a mortar and pestle.

Multi-Walled Carbon Nanotube (MWCNT)/Graphite/Epoxy Composite withn-butyl-ferrocene

A MWCNT/graphite/epoxy composite containing n-butyl-ferrocene wasprepared by combining, in dichloromethane, 17 mg of multi-walledbamboo-type MWCNTs (bamboo type 5 to 20 μm in length and having an outerwall diameter of 30+/−15 nm) (Nanolab, Brighton, Mass.) with 17 mg ofgraphite, 20 mg of n-butyl-ferrocene, 200 mg of epoxy A and 30 mg ofepoxy B. The solvent was then evaporated at room temperature.

Room Temperature Ionic Liquid RTIL Phase [C₄mpyrr][N(Tf)₂]

As described below, [C₄mpyrr][N(Tf)₂] was used as the RTIL phase inseveral of the exemplary embodiments.

1% n-butyl-ferrocene in N-butyl, N-methyl-Pyrrolidiniumbis(trifluoromethylsulfonyl)imide ([C4mpyrr][N(Tf)₂])

A solution of 1% volume:volume n-butyl-ferrocene was prepared by mixing10 uL of n-butyl-ferrocene in 1.0 mL of [C₄mpyrr][N(Tf)₂].

Materials and Methods: Electrolytic Layers

2% High Molecular Weight (HMW) Hydroxyethylcellulose Gel with 1M KCl

100 mg of high molecular weight hydroxyethylcellulose was dissolved in 5mL 1M KCl by stifling the solution over heat (approximately 80° C.)until the hydroxyethylcellulose was completely dissolved.

K₄Fe(CN)₆ in 1M KCL

0.755 g of KCl was added to 0.042 g of K₄Fe(CN)₆ in 10 mL de-ionizedwater and mixed by stifling.

Materials and Methods: Analyte Insensitive Electrode (AIE) Construction

A hollow cylindrical housing 3 inches in length and having an innerdiameter of 0.140 inches through and a counter-bore of 0.188 inches indiameter and 0.275 inches in depth, was machined from a cast PEEKpolymer rod. An internal lip was thereby formed at the point of diameterchange. A 20 gauge copper lead soldered to a circular brass disc havingthe same diameter as the housing's larger internal diameter was thenplaced within the housing, seated on top of the internal lip. An AIEcavity and a smaller-diameter rear housing cavity were thereby formed,with the brass disc forming the rear wall of the AIE cavity, and thelead extending through the rear bore and protruding externally.

The internal components of the AIE were then assembled. A conductiveplug, optionally with a RAM dispersed in it, was first formed within theAIE cavity, on top of the brass plate opposite the brass lead, and flushwith the sample end of the housing, by packing the AIE cavity with oneof the uncured conductive composite materials described above. Aftercuring (by baking at 150 degrees C. for 1 hour), the exposed end of theplug was cleaned and polished. A polymeric collar having an internaldiameter identical to the external diameter of the housing was thenfitted over the sample end of the housing, thereby elongating the AIEcavity in which the AIE phase layers were placed.

A first PEEK washer having an internal diameter of 3/16 inch and anouter diameter identical to the internal diameter of the AIE cavity wasthen placed in the AIE cavity and tamped until flush with the plug,forming a well. A drop of RTIL phase was then deposited in the well,followed by a polyethersulfone membrane (i.e. the conductive physicalbarrier) having the same diameter as the washer, and a second PEEKwasher placed on top of the membrane.

Optionally, the plug is recessed, and the PEEK washer adjacent to theplug is omitted. The RTIL phase is layered directly on the plug surface.A polyethersulfone membrane is then placed over the end of the AIEcavity.

Optionally, an additional electrolytic layer which may optionallycomprise an RAM dispersed in it is interposed between the RTIL (i.e. thefirst electrolytic layer) phase and the sample, by placing one drop ofthe second electrolytic layer solution in the well formed by the washeradjacent to the composite plug, followed by a polyethersulfone membraneand another PEEK washer.

In other embodiments, a second RTIL phase layer (i.e a secondelectrolytic layer) which may optionally comprise a RAM dispersed in itis interposed between the first electrolytic layer and the compositeplug by placing one drop of the second RTIL phase in the well formed bythe washer adjacent to the composite plug, followed by apolyethersulfone membrane and a PEEK washer.

After assembly, the layers were compressed by placing the probelengthwise in a C-clamp and exerting sufficient pressure to bring theend of the electrode collar and the contents of the housing flush withthe edge of the housing's sample.

Test Protocol

Square wave or cyclic voltammetric measurements were made using astandard three-electrode configuration using an ECHOCHEMIE AUTOLABpotentiostat/galvanometer (model PGSTAT12) with the AIE acting as theworking electrode, a saturated calomel electrode functioning as areference, and a graphite rod serving as a counter-electrode. The sampleend of the AIE, the sensor end of the calomel reference, and thecounter-electrode were placed in contact with a buffer solution ofdefined pH. The system was then subject to oxidative or reductive cyclesof square-wave voltammetry over a range of potentials and the resultingcurrent through the AIE was measured as a function of the appliedpotential.

EXEMPLARY EMBODIMENTS First Exemplary AIE Embodiment

A first exemplary AIE having a second electrolytic layer with the RAM inthe RTIL phase (i.e. the first electrolytic phase) was constructed asdescribed in the materials and methods section above using agraphite/epoxy plug, 1 drop of 1M KCl for the second electrolytic phaseand 1 drop of a 1% volume:volume solution of n-butyl-ferrocene in[C₄mpyrr][N(Tf)₂]for the RTIL phase.

Second Exemplary AIE Embodiment

A second exemplary AIE having an aqueous electrolytic phase and with theRAM in the RTIL phase was constructed in the same manner as the first,with the exception that one drop of 1M KCl in 2% HMWhydroxyethylcellulose gel was used for the second electrolytic phase.

Third Exemplary AIE Embodiment

A third exemplary embodiment having an aqueous electrolytic phase andwith the RAM in the RTIL phase was constructed generally as described inthe Materials and Methods section above, using aMWCNT/graphite/epoxy/n-butyl-ferrocene plug prepared with 17 mg MWCNT,17 mg graphite, 200 mg epoxy A, 30 mg epoxy B and 20 mg ofn-butyl-ferrocene. The reagents were combined by stifling in methylenechloride and then evaporating the solvent at room temperature. 1 drop ofaqueous 1M KCl was used for the second electrolytic layer, and 1 drop of1% volume:volume n-butyl-ferrocene in [C₄mpyrr][N(Tf)₂ ] for the RTILphase (i.e. the first electrolytic layer). This example is depicted asConfiguration 4 in FIG. 2 wherein the RAM is in both the carbon plug andthe RTIL.

Square wave voltammetric measurements were then made from the thirdexemplary embodiment as described in the test protocol section above,for a total of 7,000 continuous oxidative cycles of square wavevoltammetry over a potential range from negative 0.8 to positive 0.8volts, and the resulting current through the AIE as a function of theapplied potential was measured. The results are shown in FIGS. 5A and5B. In this experiment, 1000 scans=430 minutes.

A control experiment was also performed using the construct andmaterials and square wave measurements described for the third exemplaryembodiment with the exception that the electrolytic layers were absentin the construct. FIG. 5C is a plot showing the results from thiscontrol experiment in a plot overlayed with the data from the first 2000scans of the experiment described in the preceding paragraph.

One can see from the data shown in FIG. 5C that the signal derived fromthe electrode without electrolytic layers in the construct drifts 28 mVin the first 258 minutes (600 scans) (see FIG. 5C) while thecorresponding signal derived from the electrode employing theelectrolytic layers remained much more stable (drifting less than 5 mV)over the same period of time (see FIG. 5C).

Fourth Exemplary AIE Embodiment

A fourth exemplary AIE having an aqueous electrolytic layer with one RAMin the aqueous electrolytic layer and another, different RAM in the RTILphase was constructed using the same housing and graphite/epoxy plug aswere used in the first embodiment. 1 drop of a solution of 1M KCl and 10mM K₄Fe(CN)₆ was used for the second electrolytic layer, and 1 drop of1% volume:volume n-butyl-ferrocene in [C₄mpyrr][N(Tf)₂] was used for theRTIL phase.

Fifth Exemplary AIE Embodiment

A fifth exemplary embodiment without a second electrolytic phase butcontaining a RAM in the composite plug was constructed generally asdescribed in the materials and methods section above using aMWCNT/graphite/epoxy/n-butyl-ferrocene plug prepared by combining 30 mgof n-butyl-ferrocene, 49 mg MWCNT, 49 mg graphite, 575 mg epoxy A and 56mg epoxy B in methylene chloride, stifling, then evaporating the solventat room temperature. The plug was recessed slightly in the housing. Nowasher was used adjacent to the plug; instead, 1 drop of[C₄mpyrr][N(Tf)₂] deposited directly on the recessed plug was used asthe RTIL electrolytic layer phase. A polyethersulfone membrane was thenused as the conductive physical barrier and layered directly over theRTIL phase, followed by a PEEK washer. This is depicted as configuration1 in FIG. 2.

Square wave voltammetric measurements were then made from the fifthexemplary embodiment as described in the test protocol section above,for a total of 20 continuous oxidative cycles of square wave voltammetryover a potential range of −0.8 to +0.8V and the resulting currentthrough the AIE as a function of the applied potential was measured. Themeasurements were made at pH 2, 4, 7, 8, 9.18 and 10 using buffershaving compositions shown in Table 1 and solutions of 10 mM HCl and 1000mM KCl. The results are shown in FIGS. 6A and 6B.

A control experiment was also performed using the construct andmaterials and square wave measurements described for the fifth exemplaryembodiment with the exception that the RTIL electrolytic layer phase andpolyethersulfone membrane were absent in the construct. FIG. 6C showsthe results from this control experiment.

One can see by comparing the results in FIG. 6C and FIG. 6D that thepresence of the electrolytic layer (in this case the RTIL) is effectivein increasing the stability of the AIE signal across a range ofsolutions with various compositions. Without the electrolytic layer thesignal varied across the samples by 84 mV. While using the AIEconstruct, the signal varied only 16 mV.

TABLE 1 Test Buffer Compositions pH 2 pH 4 pH 7 pH 8 pH 9.18 pH 10glycine potassium dibasic dibasic sodium sodium HCl acid sodium sodiumborate bicarbonate phthalate phosphate phosphate decahydrate sodiummonobasic monobasic carbonate potassium potassium phosphate phosphate

Sixth Exemplary AIE Embodiment

A sixth exemplary embodiment without a second electrolytic layer andhaving the RAM in the plug was constructed with aMWCNT/graphite/epoxy/plug. The PEEK washer adjacent to the plug wasomitted; instead, a polyethersulfone membrane wetted with 1%volume:volume solution of n-butyl-ferrocene in [C₄mpyrr][N(Tf)₂] wasplaced on top of the plug, followed by a PEEK washer, and the electrodewas then compressed as described above. This embodiment is depicted inFIG. 7.

These and other embodiments of the invention are provided forillustration and not limitation of the various aspects and embodimentsof the invention set forth in the following claims.

What is claimed is:
 1. A multi-phase analyte insensitive electrode (AIE)for use in an electrochemical sensing device for measuring an analyte ina sample, the AIE comprising (a) an electrolytic layer; (b) anelectrically conductive component electrically connected to theelectrolytic layer; (c) a redox active material capable of being atleast one of electrically oxidized and electrically reduced, the redoxactive material being dispersed in at least one of the electrolyticlayer and the conductive component, the redox active material beingselected from the group consisting of n-butyl-ferrocene, silvernanoparticle-coated glassy carbon, ferrocene, polylvinylferrocene,NiHCF, ferrocene styrene copolymers, ferrocene styrene cross-linkedcopolymers, Ni Cyclam, and K₄Fe(CN)₆, anthraquinone (AQ), anthracene,9,10-phenanthrenequinone (PAQ), 1,4-benzoquinone, 1,2-benzoquinone,1,4-napthaquinone, 1,2-napthaquinone, N,N′-diphenyl-p-phenylenediamine(DPPD), azo containing compounds, azobenzene and derivatives thereof,porphyrins and derivatives thereof, octaethylporphyrin,tetraphenylporphyrins, metalloporphyrins and derivatives thereof, hemin,iron octaethylprophyrin, iron tetraphenylporphyrin, and viologens andderivatives thereof, and methyl viologen; and (d) a conductive physicalbarrier positioned so that it is interposed between the electrolyticlayer and a sample when the electrode is in contact with the sample, andthat physically separates the electrolytic layer from the sample.
 2. TheAIE of claim 1, wherein the electrolytic layer is selected from thegroup consisting of: (i) an ionic liquid (IL) comprising an anionicchemical species and a cationic chemical species; (ii) a fluorous phasecomprising a fluorous organic liquid and a dispersed electrolyte; (iii)an organic phase comprising an organic liquid and a dispersedelectrolyte; and (iv) an aqueous phase comprising water and a dispersedelectrolyte.
 3. The AIE of claim 2, wherein the electrolytic layer is atleast one of an ionic liquid (IL) and a room temperature ionic liquid(RTIL).
 4. The AIE of claim 1, wherein the redox active material isselected from the group consisting of a redox-active organic molecule,redox-active polymers, metal complexes, organometallic species, metals,metal salts, or semiconductors, and wherein the redox active materialundergoes one or more electron transfer processes.
 5. The AIE of claim1, wherein the conductive component comprises an electrically conductivematerial selected from the group consisting of carbon allotropes andderivatives thereof, transition metals and derivatives thereof,post-transition metals and derivatives thereof, conductive metal alloysand derivatives thereof, silicon and derivatives thereof, conductivepolymeric compounds and derivatives thereof, and semiconductor materialsand derivatives thereof.
 6. The AIE of claim 1, wherein the conductivecomponent further comprises a composite material comprising a binder andan electrically conductive material.
 7. The AIE of claim 6, wherein theelectrically conductive material comprises at least one of graphite,glassy carbon, multi-walled carbon nanotubes (MWCNTs), single-wallednanotubes (SWNTs), boron-doped diamond, and any combination thereof. 8.The AIE of claim 7, wherein the composite material further comprises theredox active material.
 9. The AIE of claim 1, wherein the conductivephysical barrier is selectively impermeable to an analyte or tonon-analyte species in the sample or both.
 10. The AIE of claim 1,wherein the conductive physical barrier is selected from the groupconsisting of a porous frit, a film, a microporous membrane, and anonporous membrane comprising a solid solution of IL in a polymeric orinorganic material.
 11. The AIE of claim 1, wherein the electrolyticlayer is the room temperature ionic liquid (RTIL) N-butyl-N-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide ([C₄mpyrr][N(Tf)₂]);the electrically conductive component is composed of multi-walled carbonnanotubes, graphite, and epoxy; the redox active material (RAM) isn-butyl ferrocene; and the conductive barrier layer is apolyethersulfone membrane in direct contact with and thus saturated bythe RTIL.
 12. The AIE of claim 1, further comprising a secondelectrolytic layer.
 13. The AIE of claim 1, further comprising: (e) asecond electrolytic phase adjacent to the first electrolytic layer andsubstantially immiscible with the first electrolytic layer, wherein thesecond electrolytic phase is interposed between the conductive componentand the first electrolytic layer and is in electrical connection withthe first electrolytic layer and the conductive component.
 14. The AIEof claim 12, further comprising: (f) a second electrolytic phase with aconductive physical barrier interposed between the second and the firstelectrolytic layers, wherein the redox active material is optionallydispersed in the second electrolytic layer and wherein the secondelectrolytic phase is interposed between the conductive component andthe first electrolytic layer and is in electrical connection with thefirst electrolytic layer and the conductive component.
 15. The AIE ofclaim 13, further comprising a second electrolytic layer.
 16. The AIE ofclaim 15, wherein the second electrolytic layer is selected from thegroup consisting of an aqueous electrolyte solution, a gelled aqueouselectrolyte solution, a electrolytic sol gel, an ionic liquid, aelectrolyte containing fluorous layer, and an organic electrolytesolution.